响应面法优化考马斯亮蓝G-250溶液的配制

Optimization of Brilliant Blue G-250 Dye Solution Using Response Surface Methodology

试验研究了各组分磷酸、乙醇、考马斯亮蓝G-250在考马斯亮蓝G-250溶液中的作用及其对测定蛋白质浓度灵敏度和准确度的影响,结合全波长扫描光谱法分析了溶液配制过程中的变色机理。在此基础上,以一系列牛血清白蛋白溶液标准浓度和测定值之间的差值平方和的均方(S值)为响应值,采用单因素试验和响应面分析法优化考马斯亮蓝G-250溶液的配制条件。结果表明,乙醇是用来降低水溶液的极性环境,促进考马斯亮蓝G-250的溶解;磷酸是为考马斯亮蓝G-250表面电荷发生变化的促进剂;两者构成了影响变色灵敏度的主要因素,考马斯亮蓝G-250分子在溶液中不同的存在形式使反应体系呈现出从蓝色→绿色→棕红色等颜色变化;响应面优化配制条件为:60 mg/L考马斯亮蓝G-250、50 mL/L 95%乙醇、130 mL/L 85%磷酸时,S值最小值为2.07,即在此配制条件下的牛血清白蛋白测定值与标准值最接近。同时以不同标准蛋白质为试样对所配制的溶液进行准确度和灵敏度试验,结果表明对牛血红蛋白、γ-球蛋白测定准确度和灵敏度较好,对胰蛋白酶、胃蛋白酶灵敏度较差,主要原因是由于蛋白质组成差异所引起的。

Phosphoric acid, ethanol and Coomassie brilliant blue G-250 were respectively studied to explain how to influence theaccuracy and the sensitivity on protein assay. The mechanism of color changing during the preparation of the dye solution was studied with UV spectrophotometer. The brilliant blue G-250 Dye solution was optimized by mono-factors experiments and response surface methodology. The results showed that ethanol is used to provide polar environment for Coomassie brilliant blue G-250 dissolution, phosphoric acid is the accelerator for Coomassie brilliant blue G-250 surface charge, constituting the main factors that affect the color sensitivity. Coomassie brilliant blue G-250 reagent in the reaction system showed a color change such as blue→green → red and other color changes, mainly due to the different aggregation levels of the polymers formed by hydrophobic interactions between molecules Coomassie brilliant blue G-250. The quadratic regression analysis was conducted on the optimized results by using DesignExpert software. It was found that the optimum conditions for brilliant blue G-250 Dye solution were as follows ：60 mg/L Coomassie brilliant blue G-250,50 mL/L 95% ethanol and 130 ml_,/L 85% phosphoric acid. Under this condition, the value S was 2.07. It meant that the determination value was closer to the true value of bovine serum albumin. And the other different standard protein samples were tested. The results showed that the measurement accuracy and the sensitivity of Bovine Hemoglobin, γ-globulin were better than trypsin, and pepsin mainly due to different compositions in protein.